Proteases participate in a variety of developmental and metabolic processes (Stroud R, Sci. Am. 231:74-88 (1974) and Neurath H, Science 224:350-357 (1984). Molecular defects that alter enzyme function often lead to serious human diseases, such as bleeding, thrombosis, and atherosclerosis. Hepsin is a novel serine protease of the trypsin family and contains a transmembrane domain near its amino-terminus, Kurachi et al., Methods in Enzymology, 244:100-114 (1994). This structural feature distinguishes hepsin from most other serine proteases. In the fruitfly Drosophila melanogaster, another transmembrane serine protease, Stubble-stubbloid, has been reported that has similar structural features as that of hepsin. Stubble-stubbloid protein plays an important role in epithelial morphogenesis and development in Drosophila. Defects in Stubble-stubbloid gene cause malformation of legs, wings and bristles, Appel et al., Proc. Natl. Acad. Sci. USA 90:4937-4941 (1993).
The human hepsin cDNA was initially isolated from a liver cDNA library screened with a mixture of oligonucleotides based on a consensus sequence of serine proteases, Leytus et al., Biochemistry 27:1067-1074 (1988). Biochemical studies indicate that hepsin is a type II transmembrane serine protease expressed mainly on the surface of hepatocytes, Tsugi et al., J. Biol. Chem. 266:16948-16953 (1991). Lower levels of hepsin mRNA are detected in other tissues including lung, kidney, pancreas, stomach, thyroid and prostate. In addition, hepsin mRNA is present in several human tumor cell lines, such as hepatoma cells HepG2 and PLC/PRF/5, mammary cancer cells MCF784 and T470, and epitheloid carcinoma cells HeLa S3, Tsuji et al., J. Biol. Chem. 266:16948-16953 (1991) and Torres-Rosado et al., Proc. Natl. Acad. Sci. USA 90:7181-7185 (1993).
Hepsin has a number of reported activities. In an in vitro study, recombinant human hepsin expressed on the cell surface activated blood coagulation factor VII but not factors IX, X, prothrombin or protein C, all of which share significant structural and sequence similarities with factor VII. The activation of factor VII by hepsin was shown to be sufficient to initiate the coagulation pathway leading to thrombin formation, Kazama et al., J. Biol. Chem. 270:66-72 (1995). Elevated plasma factor VIIa activity has been known to be a significant risk factor for ischemic heart disease and cardiovascular death, Hultin, Prog. Hemostasis Thrombosis 10:215-241, (1991) and Mann, Arteriosclerosis 9:783-784 (1989). Factor VIIa/tissue factor complex also contributes to tumor-related hypercoagulability and intravascular thrombosis, Edwards et al., Thromb. Haemostasis 69:205-213 (1993).
In addition to blood coagulation, hepsin was reported to be critical for cell growth. In a cell culture system, addition of anti-hepsin antibodies or hepsin-specific antisense oligonucleotides to the culture medium significantly inhibited growth of hepatoma cells Torres-Rosado et al., Proc. Natl. Acad. Sci. USA, 90:7181-7185 (1993). This observation is quite interesting in light of the expression of hepsin mRNA in a number of tumor cells. The growth factor-like activities of serine proteases have been known for many years. For example, thrombin is a potent mitogen for vascular fibroblasts and smooth muscle cells, Fenton J, Ann. N.Y. Acad. Sci., 485:5-15 (1986). Furthermore, serine proteases also participate in processing of growth factors, Massague J, J. Biol. Chem., 265:21393-21396 (1990). The hepsin-dependent tumor cell growth indicates a mechanism in which hepsin functions either directly as a growth factor or indirectly as an enzyme that processes certain growth factors essential for cell growth.